GSM7838114: BW_F_15504_midbrain; Peromyscus maniculatus; RNA-Seq
For the whole brain dataset, a cohort of 59–137-day-old virgin male P. maniculatus (n=5) and P. polionotus (n=6) animals were euthanized by CO2 inhalation 7–10 h before the start of the dark period. Whole brain samples were then rapidly dissected, flash-frozen, and stored at -70°C. We homogenized the samples in TRIzol Reagent (Invitrogen, 15596026) using a Bio-Gen PRO200 homogenizer (PRO Scientific, Oxford, USA), and extracted RNA using a Direct-zol™ RNA MiniPrep Plus kit (Zymo Research, R2070), including a DNase I treatment; For the brain subregion dataset, a cohort of 82–85-day-old virgin male and female P. maniculatus, P. polionotus, and their F1 hybrids were euthanized by CO2 inhalation followed by cervical dislocation 3–10 h before the onset of the dark period. One cohort was used for dissection of the cerebellum, hippocampus, and hindbrain samples. In these samples, after the brain was removed, a dorsal-to-ventral incision was made posterior to the cerebrum to release the hindbrain and cerebellum. The cerebellar peduncles were cut, and the hindbrain and cerebellum were placed in tubes and flash frozen in liquid nitrogen. The petri dish with the remaining brain was then placed on ice, and the two cerebral hemispheres were cut apart. For each hemisphere separately, the hippocampus was severed from the dorsal fornix and gently scooped out of the hemisphere with a brush in ventral-to-dorsal direction (Lu, Airey, and Williams 2001), and then flash frozen. For a second cohort of animals, we dissected the brains and then immediately submerged them into embedding compound for cryosectioning (Tissue-Tek O.C.T.) and froze them on dry ice. We sectioned brains on a coronal plane at 150 μm using a cryostat at -20°C (Leica CM3050 S, Leica Biosystems Inc., Buffalo Grove, IL) and stored the sections at -70°C until we dissected the medial prefrontal cortex, septum, striatum, hypothalamus, amygdala, thalamus, and midbrain with sample corers according to anatomical landmarks. The sample cores were immediately transferred into homogenization buffer. After subregion dissection, we homogenized samples for 10 seconds in Maxwell RSC Homogenization Buffer (Promega, Madison, USA) with 2% 1-thioglycerol using a Bio-Gen PRO200 homogenizer (PRO Scientific, Oxford, USA) and then extracted RNA using the Maxwell RSC simplyRNA Tissue Kit (Promega). We prepared each whole brain sequencing library from 1 μg of total RNA using a PrepX PolyA mRNA Isolation Kit (Wafergen, 40047) on the Apollo 324 NGS Library Prep System (Takara Bio USA Holdings, Mountain View, CA). We added Illumina indices and amplified libraries with 12 cycles of PCR and cleaned them using PCRClean DX beads (Aline Biosciences, Woburn, MA); We prepared brain subregion RNA-Seq libraries using an mRNA HyperPrep kit (Kapa Biosystems, Wilmington, USA) automated on a BioMek FXp (Beckman Coulter, Brea, USA). Post-capture, we fragmented mRNA for 6 min at 94°C for a target insert size of 200-300 base pairs. We amplified dual-indexed adapter-ligated libraries in 11 PCR cycles.